The in vivo synthesis of plant sesquiterpenes by Escherichia coli

Three plant genes encoding (+)-delta -cadinene, delta -epi-aristolochene, and vetispiradiene cyclases were expressed in Escherichia coli to evaluate the potential of this bacterium to synthesize sesquiterpenes in vivo, Various growth temperatures, carbon sources, and host strains were examined to optimize terpene production. The highest levels of sesquiterpene production occurred when the enzymes were expressed in strain DH5 alpha from the trc promoter (Ptrc) of the high-copy plasmidpTrc99A in M9 medium supplemented with 0.2% (v/v) glycerol at 30 degreesC for delta -epi-aristolochene and vetispiradiene and 37 degreesC for (+)-delta -cadinene. The highest concentrations of sesquiterpenes observed were 10.3 mug of (+)-delta -cadinene, 0.24 mug of delta -epi-aristolochene (measured as (+)-delta -cadinene equivalents), and 6.4 mug of vetispiradiene (measured as (+)-delta -cadinene equivalents) per liter of culture. These sesquiterpene production levels are > 500-fold lower than carotenoid production, both of which are synthesized from endogenous trans-farnesyl diphosphate (FDP) in E. coli. Based on these results, we conclude that the limiting factor for sesquiterpene synthesis in E. coli is the poor expression of the cyclase enzyme and not supply of the FDP precursor. (C) 2001 John Wiley & Sons, Inc.